DNA damage response (DDR) acts as a barrier to tumorigenesis. DDR contributes to the maintenance of genome integrity. In response to DNA damage, cells activate cell cycle checkpoints or apoptosis to allow repair or remove damaged cells thereby maintaining genome integrity. Checkpoint kinase 2 (Chk2), an effector kinase plays a central role in the checkpoint response and apoptosis in the DDR. Chk2 is believed to be a multiorgan cancer susceptibility gene that increases cancer risk with other factors. Extensive analyses of Chk2 and Rad53 (yeast Chk2 ortholog) have highlighted the important roles of Chk2 in S phase. Rad53 regulates DNA replication through its functions in transcription, inhibition of origin firing and stabilization of replication fork. Animal experiments also showed that loss of Chk2 in conjunction with mutations of genes of the DDR pathway where S and G2/M checkpoints are compromised, increases intra S phase checkpoint defects and promotes tumorigenesis in mice. These studies have led to the paradigm for the critical tumor suppressor function of Chk2 in response to DNA damage associated with replication. Objectives I hypothesize that Chk2 suppresses oncogenicity caused by replication stress and I will study the mechanisms underlying the tumor suppressor role of Chk2 in response to replication associated DNA damage. Methods For attempts to identify new Chk2 substrates, I have performed SILAC (Stable Isotope Labeling with Amino acids in Cell culture) screening using cells with analog-sensitive Chk2 allele. This large-scale phosphoproteomic screening has resulted in the finding of new Chk2 targets, minichromosome maintenance (MCM) that might be associated with Chk2 functions in S-phase. I will study the role of Chk2 in the DDR especially in S-phase, using non-transformed conditional knockout cells that will be generated by somatic gene targeting. Using this cell line, I will analyze the acute Chk2 deficiency phenotypes and perform structure- functional analysis to dissect Chk2 functions based on the phenotypes examined. Further, I will study the MCM complex as Chk2 target using mammalian and yeast systems to uncover the mechanisms underlying the tumor suppressor role of Chk2 in replication. Aim 1. Generation of Chk2 knockout cell lines by using somatic gene targeting approach Aim 2. Determine the role of Chk2 at the cellular level 1.1 Phenotypic analysis of Chk2 deficient cells 1.2 Structure-functional analysis of Chk2 Aim 3. Determine the role of Chk2 in the regulation of MCM complex function 2.1 Identification of MCM3 as a Chk2 target 2.2 Role of MCM3 phosphorylation by Chk2 2.3 Functional analysis of MCM3 phosphorylation in yeast Closing Remarks Understanding the roles and mechanism of Chk2, a protein that has important functions in tumor suppression, will provide a novel therapeutic strategy to improve current therapeutic methods.